1. Field of the Invention
The present invention relates to a method for detecting and identifying a desired specific base sequence in a nucleic acid (DNA or RNA) of viruses, animals, plants, and human beings, or detecting mutation in a base sequence. The present invention also relates to a probe for the above method.
2. Related Background Art
As the result of progress of nucleic acid analysis technique, a number of mutant genes have been found, and a variety of genetic diseases caused by a mutant gene are being elucidated. Some of the genetic diseases have been found to be caused by local deficiency of a base, or point mutation of a base in a gene. The abnormality in the gene gives rise to mutation of protein to show various symptoms. At present, such genetic diseases are diagnosed mainly after manifestation of the symptom by enzymatic assay or by an immunological method employing an antibody. However, from the standpoint of early therapy, it is considered to be important to find mutation of the gene before manifestation of a serious symptom.
One of effective methods for the diagnosis is RFLP (restriction fragment length polymorphism). In this method, for example, the whole gene of a human being is cut by restriction enzymes, and the resulting DNA fragment is developed by agarose gel electrophoresis, fixed on a filter by a Southern blotting method, and hybridized with a probe comprising a DNA (or RNA) labeled by an isotope or the like. From the difference of the cut pattern of the DNA of the sample from that of a normal DNA, the gene is detected which causes the disease.
The DNA diagnosis is useful not only for genes of human being but also for identification of infecting bacteria.
Hitherto, the kind of an isolated bacterium is identified by similarity in morphological properties and biochemical properties thereof. This method has disadvantages in that incubation of the bacterium requires a long time, judgment of the properties depends on the method of testing, the identification result differs depending on the selection of properties to be tested, and so forth.
In recent years, DNA-DNA hybridization or DNA-RNA hybridization has been tried particularly in detection and identification of microbism-causing bacteria. In this method, nucleic acid (DNA or RNA) is extracted from a bacteria, and a specified portion of nucleic acid of the bacteria which has a base sequence having homology to the tested nucleic acid sample is detected by hybridization, whereby the presence of the targeted bacteria in the sample is judged.
The hybridization, which is a basic technique for the above test, generally comprises the steps below.
(1) Cutting of DNA into fragments, and development thereof by gel-electrophoresis; PA0 (2) Adsorption of the developed respective DNA fragments on a nitrocellulose filter (Southern blotting); PA0 (3) Formation of a hybrid by reaction of the DNA fragment on the nitrocellulose filter in the step (2) above with a probe; and PA0 (4) Detection of the DNA fragment which has formed a hybrid.
In hybridization between DNAs, a labeled probe DNA and a targeted DNA form a hybrid between the respective complementary portions by hydrogen bonding.
The probes employed in the hybridization reaction are changing with the times. In the earliest stage of the research, a long DNA fragment was labeled with an radioactive isotope by nick translation. With the development of a DNA synthesizer, a synthesized oligonucleotide has been used in place of a long DNA, and the labeling substance has been changed from a dangerous radioactive isotope to a safe biotin-avidin type reagent, and further to a chemiluminescence type reagent.
For precise hybridization between complementary sequences, the reaction temperature and the ion strength should be selected to be optimum. At a higher temperature, the probe does not combine with the nucleic acid having a complementary sequence. At a lower temperature, the probe combines non-specifically with the nucleic acid. For higher preciseness, it is necessary to eliminate instable hydrogen bonding by lowering the salt concentration of the solution or raising the temperature of the solution and to wash away non-specifically combined probes or mismatched probes. Accordingly, many trial-and-error experiments are required to select the optimum conditions for the reaction and the washing.
In genetic diagnosis, for higher precision, the conditions for the hybridization reaction and the washing should be decided strictly such that mismatch of one base pair level is eliminated.
In a hybridization reaction, immobilization of a targeted nucleic acid on a support like nitrocellulose has the advantage of ease in washing for elimination of non-specific bonding, etc. of the probe, but has disadvantages in complexity of operation, difficulty in automation of the test, and an extended time required for operation. Therefore, this method is not suitable for treatment of a large number of samples.
If a method is found for detecting a hybrid in a solution without immobilization of nucleic acid, it enables automation of the detection. Many attempts are being made therefor. The most important problem in eliminating the nucleic acid immobilization step is how to differentiate the probe combined with the targeted nucleic acid from excess non-combined probe (namely B/F separation). In this method also, selection of the reaction conditions and the washing conditions is important similarly as in the aforementioned hybridization employing immobilized nucleic acid to avoid non-specific adsorption and mismatch of probes.
To detect a hybrid of targeted nucleic acid with a probe without B/F separation, several methods are disclosed which employ fluorescence depolarization (see Japanese Patent Application Laid-Open Nos. 2-295496 and 2-75958). In these methods, a fluorescence-labeled single-stranded DNA probe is brought into contact with a DNA in a sample to form a double-stranded DNA, and the change of the fluorescent polarization by the double strand formation is measured. Thereby the presence of a base sequence in the DNA in the sample corresponding that of the probe is detected. Such a method is grounded on the principle that a fluorescent substance bonded to the single strand is made less movable by formation of a double strand to increase its fluorescence anisotropy.
In these methods, however, a complicated preliminary operation is necessary to remove completely any contaminant such as protein from the sample, since a contaminant in the sample will be adsorbed non-specifically by the probe DNA to increase background in hybrid detection. Further, non-specifically adsorbed probe DNA and a pseudo-hybrid formed by mismatch with a base need to be removed similarly as in detection in other solution systems. In these methods, although B/F separation is not necessary, the probe concentration should be at the same level as that of the targeted DNA in measurement of the change of fluorescent polarization.
As described above, the detection of a targeted nucleic acid by hybridization reaction, when the B/F separation is required for the detection, is troublesome because of a number of operations such as B/F separation (removal of excess probe), removal of non-specifically adsorbed matter and mismatching probes, and so forth, whether the targeted nucleic acid is immobilized or not. Furthermore, the optimum conditions of the respective operations vary depending on the probe length, and the respective base sequences, so that the operating conditions have to be set for each of the cases. In particular, the location of mismatched base on the probe affects significantly the stability of the hybrid, and in some cases the mismatched hybrid is not removable owing to the location of the mismatching base. Therefore, the conditions for hybridization reaction have to be decided in consideration of the liability of mismatching of the base, which requires further troublesome operations.
The detection methods by measurement of change of fluorescence without B/F separation also require complicated treatment for preventing non-specific adsorption and mismatching, or for removing non-specifically adsorbed matters or mismatched matters. Moreover, a contaminant may impair the sensitivity of the measurement, and the probe concentration needs to be at the same level as the targeted nucleic acid. Therefore, these methods require use of a sufficient amount of a sample, and cannot be applied to microanalysis, disadvantageously.
The present invention has been made to cancel the aforementioned disadvantages of prior art.